PROJECT SUMMARY There is a strong genetic component to asthma and numerous genome-wide association studies (GWAS) have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma in a number of ethnically diverse populations. However, how ORMDL3 contributes to asthma pathogenesis and its functions in the lung are not well understood. Our published and preliminary data uncovered the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis. Our data suggest that physiologically, ORMDL3 is a negative regulator of serine palmitoyltransferase in de novo ceramide biosynthesis; remarkably however, highly elevated pathological ORMDL3 expression, such as observed in allergic asthma, enhanced ceramide levels mainly due to increased recycling/salvage pathway of sphingolipid degradation. In turn, elevated ceramide exacerbated inflammatory responses. We found that allergen challenge increased expression of ORMDL3 in mice and concomitantly increased ceramide in lung and bronchoalveolar lavage fluid. Nasal administration of the immunomodulator FTY720 reduced both ORMDL3 expression and ceramide production, mitigating airway inflammation and hyperreactivity (AHR). Ceramide levels were also increased in exhaled breath condensates from a small cohort of asthmatic patients. Hence, we suggest that ceramide is a new critical player in allergic asthma exacerbation. Our overarching hypothesis is that lung epithelial expression of ORMDL3 is greatly increased during Th2-mediated allergic asthma leading to an elevation of ceramide that is critical for induction of AHR and the exacerbation of allergic inflammation. We propose that ceramide functions through several non-mutually exclusive mechanisms and that ceramide itself may be a viable therapeutic target and a biomarker for allergic asthma. To test our central hypothesis, in Aim 1 we will determine how elevated levels of ceramide contribute to allergic lung inflammation in mice. In Aim 2, we will investigate strategies to target ceramide and explore the cell autonomous role of ORMDL3 and dysregulation of ceramide in allergic asthma. As additional characterizations of cohorts of asthmatics are needed to better understand the different phenotypes leading to more personalized targeted approaches of asthma therapy in Aim 3, we will examine the potential of ceramide as a biomarker for atopic asthma. Our proposed studies will help to establish the pathological function of the ORMDL3-ceramide axis, link enhanced ceramide levels to asthma severity, identify a new player in asthma pathogenesis, and demonstrate that novel strategies targeting ceramide could be a promising new therapeutic approach.